1. T Cells and T Cell-related Pathologies
Immune cells respond to the presence of foreign antigens with a wide range of responses, including the secretion of preformed and newly formed mediators, phagocytosis of particles, endocytosis, cytotoxicity against target cells, as well as cell proliferation and/or differentiation. T cells are a subgroup of cells which together with other immune cell types (polymorphonuclear, eosinophils, basophils, mast cells, B cells, and NK cells), constitute the cellular component of the immune system (U.S. Pat. No. 6,057,294; US Pat. Appl. 20050070478). Under physiological conditions T cells function in immune surveillance and in the elimination of foreign antigen. However, under pathological conditions there is compelling evidence that T cells play a major role in the causation and propagation of disease. In these disorders, breakdown of T cell immunological tolerance, either central or peripheral is a fundamental process in the causation of autoimmune disease.
Central tolerance involves thymic deletion of self reactive cells (negative selection) and positive selection of T cells with low affinity for self major histocompatibility complex antigens (MHC). In contrast, there are four, non-mutually exclusive hypotheses that have been proposed to explain peripheral T cell tolerance which are involved in the prevention of tissue specific autoimmune disease. These include: anergy (loss of co-stimulatory signals, down regulation of receptors critical for T cell activation), deletion of reactive T cells, ignorance of the antigen by the immune system and suppression of autoreactive T cells. Tolerance once induced does not necessarily persist indefinitely. A breakdown in any of these mechanisms may lead to autoimmune disease (Srinivasan et al. Cytokine. 2009; 46:147-59; Mescher et al. Semin Immunol 2007; 19:153-61; US Pat. Appl. 20050070478).
Numerous diseases are believed to result from autoimmune mechanisms (Allen et al. J Pept Res 2005; 65:591-604; WO 2006077601). A non-exhaustive list of autoimmune disorders include: systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, type I diabetes, gastroenterological conditions e.g. inflammatory bowel disease e.g. Crohn's disease, primary biliary cirrhosis, chronic active hepatitis; skin problems e.g. atopic dermatitis, psoriasis, pemphigus vulgaris; cardiovascular problems e.g. autoimmune pericarditis. Autoimmune diseases affect millions of individuals worldwide and the cost of these diseases, in terms of actual treatment expenditures and lost productivity, is measured in billions of dollars annually. Millions of individuals suffer from or are affected by autoimmune diseases. Over 400 000 people in the United States suffer from multiple sclerosis (MS), while diabetes affects about 18.2 million people (6.3% of the population). Of the 18.2 million people with diabetes, 5-10% have type I (juvenile) diabetes. Over 16 000 new cases of lupus are reported every year, with the total number of diagnosed cases ranging between 500 000 and 1.5 million; and 2.1 million people (almost 1% of the American population) are affected by rheumatoid arthritis. Psoriasis affects 4.5 million people, with 23% of those further diagnosed with psoriatic arthritis. Irritable bowel syndrome/inflammatory bowel disease (IBS/IBD) affects approximately 10-20%, or up to one in five people in America. T cells also play a major role in the rejection for organ transplantation or graft versus host disease by bone marrow (hematopoietic stem cell) transplantation. Regulation of such immune responses is therefore therapeutically desired.
2. T Cell Receptor
Autoimmune disease and other T cell-related pathologies are characterised by the recruitment of T cells to sites of inflammation. At these sites, T cells, coupled with their ability to produce and regulate cytokines and influence B cell function, orchestrate the immune response and shape the final clinical outcome.
T cells respond to antigen via a polypeptide complex composed of the ligand-binding T cell receptor (TCR) disulfide-linked α and β subunits (or γ and δ subunits in γδ T cells) that have single transmembrane (TM) spans per subunit and small intracellular tails and associate non-covalently with hetero-(CD3γε and CD3δε) and homodimeric (ζζ) signaling subunits (Cambior J. C. Curr Opin Immunol 1992; 4:257-64; A. B. Sigalov, ed, Multichain Immune Recognition Receptor Signaling: From Spatiotemporal Organization to Human Disease, Springer-Verlag, New York, 2008). The CD3ε, δ, and γ chains have single Ig-family extracellular domains, single presumably α-helical TM spans, and intrinsically disordered intracellular domains of 40-60 residues, whereas each ζ subunit has a small extracellular region (9 residues) carrying the intersubunit disulfide bond, a single presumably α-helical TM span per subunit, and a large, intrinsically disordered cytoplasmic domain of ˜110 residues. An understanding of the process of TCR-mediated TM signal transduction and subsequent T cell activation, leading to T cell proliferation and differentiation, is therefore pivotal to both health and disease. Disturbance in this intricate structure-function relationship of TCR, harmonising antigen recognition with T cell activation may provide the therapeutic means to deal with inflammatory and other T cell-related disorders.
3. Viral Modulation of T Cell Receptor Signaling
To successfully infect, replicate and persist in the host, viruses have evolved numerous strategies to take control of multiple cellular processes including those that target TM signal transduction mediated by immune receptors including TCR (Jerome K. R. J Virol 2008; 82:4194-204; Sigalov A. B. PLoS Pathog 2009; 5: e1000404; Kim W. M. and Sigalov A. B. Adv Exp Med Biol 2008; 640:325-49; Sigalov A. B. Adv Exp Med Biol 2008; 640:268-311). Recent breakthroughs in our improved understanding of the TCR-targeted strategies used by the viruses to escape from the host immune surveillance reveal new therapeutic targets for antiviral as well as immunomodulatory therapy (Sigalov A. B. Adv Exp Med Biol 2007; 601; 335-44; Sigalov A. B. PLoS Pathog 2009; 5: e1000404). Therefore, further investigation of how viruses have adapted to disarm the innate and adaptive immune system will prove invaluable in rational drug design efforts aiming to reduce immune activation or inflammation. In particular, viral T cell evasion strategies can be transferred to therapeutic strategies to treat T cell-mediated diseases that require similar functionalities. Viruses represent years of evolution and the efficiency and optimization that come along with it.
4. Treatment of T Cell-related Pathologies
Traditional reagents and methods used to attempt to regulate an immune response in a patient also result in unwanted side effects and have limited effectiveness (WO 2006077601). For example, immunosuppressive reagents (e.g., cyclosporin A, azathioprine, and prednisone) used to treat patients with autoimmune diseases also suppress the patient's entire immune response, thereby increasing the risk of infection, and can cause toxic side effects to non-lymphoid tissues. Due to the medical importance of immune regulation and the inadequacies of existing immunopharmacological reagents, reagents and methods to regulate specific parts of the immune system have been the subject of study for many years.
Antibodies have been considered as clinically significant therapeutic agents for various T cell-related diseases. Traditional costimulatory blockade using antibodies or fragments of antibodies, while promising, is not without drawbacks. Disadvantages to these approaches include inherent immunogenicity, unwanted Fc signaling, as well as poor tissue penetration. There have also been some indications that immunosuppression can occur with long-term treatment (Allen et al. J Pept Res 2005; 65:591-604). The use of antibodies is suggested (U.S. Pat. No. 6,221,352) to treat autoimmune disorders such as rheumatoid arthritis. Specifically, this patent covers the administration of monoclonal antibodies, alone and/or coupled to cytotoxic or cytostatic agents. However, antibody therapy poses serious disadvantages. First, as antibodies are natural products they must be produced in cell lines or other live expression systems. This raises a that there could be contamination of antibody preparations by infectious agents such as prions or viruses. Although tight regulation and regulatory vigilance and surveillance can reduce this concern, the need for ongoing monitoring arid testing for contamination contributes to the high cost of developing and administering antibody therapies. In addition, antibody-based therapies require considerable logistical support. As antibodies are proteins, they cannot be given orally, except for those used to treat certain types of mucosal infectious diseases, and therefore, systemic administration is required. Another serious disadvantage of antibody-based therapies is the high costs of production, storage, and administration. Moreover, long infusions (i.e., for example, an hour or longer) require a hospital environment and are often associated with mild to very severe side effects. For example, (genengnews.com/articles/chitem_print.aspx?aid=1668&chid=2), in one trial, in which four patients in the U.K. were given an anticancer antibody reactive against an important T cell receptor (CD28) severe and life-threatening responses were observed; the cause is at present not understood. This makes large-scale clinical applications of a number of monoclonal antibodies with demonstrated therapeutic activity impossible or, at least, severely compromised. Fast degradation of the administered antibodies is another drawback of antibody-based therapy.
Peptides based on TCR-derived sequences for disrupting TCR function presumably by interfering with assembly have also been disclosed (WO 96/22306; WO 97/47644; US Pat. Appl. 20050070478). Despite multiple advantages of these peptides as compared to antibodies, they have relatively low efficacy in terms of inhibiting TCR, thus having a high potential for toxicity and side effects, while the primary criteria for rational design of these immunomodulatory peptides and optimizing their immunomodulatory activity have not been suggested.
Filoviral immunosuppressive peptides and modified derivatives thereof have been also disclosed (US Pat. Appl. 20070185025). Similarly to the TCR derived peptide sequences, these peptides demonstrate immunosuppressive activity at relatively high peptide doses, thus having a high potential for toxicity and side effects, while the primary criteria for rational design of these immunomodulatory peptides and optimizing their immunomodulatory activity have not been suggested.
Novel uses of peptides derived from the 33 amino acid residues-long HIV gp41 fusion peptide (FP1-33) domain, in methods for prevention or treatment of autoimmune and other T cell-mediated pathologies, have also been disclosed (WO 2006077601). The FP1-33 peptide was stated to be effective at concentrations about 100 fold lower than the peptides of the invention by Manolios (U.S. Pat. No. 6,057,294; US Pat. Appl. 20050070478). However, published and disclosed experimental data on immunomodulatory activity of this 33 amino acid residues-long peptide and its fragments (Cohen et al. Biochemistry 2008; 47:4826-33; Bloch et al. Faseb J 2007; 21:393-401; A. B. Sigalov. Faseb J 2007; 21:1633-34; author reply 1635; WO 2006077601) are discrepant (A. B. Sigalov. Faseb J 2007; 21:1633-34; author reply 1635; WO 2006077601). While full length FP1-33 variants have been disclosed as pharmaceutical compositions to treat T cell-mediated pathologies (WO 2006077601), in other publications it has been stated that in contrast to 16 amino acid residue-long N-terminal region (FP1-16) of FP1-33 and its fragments (FP1-8, FP5-13, and FP9-16), the C-terminal half of FP1-33 (FP17-32) has been found to be inactive in all assays used (A. B. Sigalov. Faseb J 2007; 21:1633-34; author reply 1635).
5. Prior Art
Prior art (U.S. Pat. No. 6,221,352) suggests to use antibodies to target specific TCRs to treat autoimmune disorders such as rheumatoid arthritis. Specifically, this patent covers the administration of monoclonal antibodies, alone and/or coupled to cytotoxic or cytostatic agents. However, antibody therapy poses serious disadvantages. First, as antibodies are natural products they must be produced in cell lines or other live expression systems. This raises a that there could be contamination of antibody preparations by infectious agents such as prions or viruses. Although tight regulation and regulatory vigilance and surveillance can reduce this concern, the need for ongoing monitoring and testing for contamination contributes to the high cost of developing and administering antibody therapies. In addition, antibody-based therapies require considerable logistical support. As antibodies are proteins, they cannot be given orally, except for those used to treat certain types of mucosal infectious diseases, and therefore, systemic administration is required. Another serious disadvantage of antibody-based therapies is the high costs of production, storage, and administration. Moreover, long infusions (i.e., for example, an hour or longer) require a hospital environment and are often associated with mild to very severe side effects. For example (genengnews.com/articles/chitem_print.aspx?aid=1668&chid=2), in one trial, in which four patients in the U.K. were given an anticancer antibody reactive against an important T cell receptor (CD28) severe and life-threatening responses were observed; the cause is at present not understood. This makes large-scale clinical applications of a number of monoclonal antibodies with demonstrated therapeutic activity impossible or, at least, severely compromised. Fast degradation of the administered antibodies is another drawback of antibody-based therapy.
Another prior art (U.S. Pat. No. 6,057,294; US Pat. Appl. 20050070478) suggests to use TCR-derived peptide sequences to treat autoimmune and other T cell-related disorders. Despite multiple advantages of these peptides as compared to antibodies, they have relatively low efficacy in terms of inhibiting TCR, thus having a high potential for toxicity and side effects, while the primary criteria for rational design of these immunomodulatory peptides and optimizing their immunomodulatory activity have not been suggested.
Yet another prior art (US Pat. Appl. 20070185025) provides uses of filoviral immunosuppressive peptides and modified derivatives thereof to treat T cell-mediated pathologies. Similarly to the TCR-derived peptide sequences, these peptides demonstrate immunosuppressive activity at relatively high peptide doses, thus having a high potential for toxicity and side effects, while the primary criteria for rational design of these immunomodulatory peptides and optimizing their immunomodulatory activity have not been suggested.
Yet another prior art (WO 2006077601) provides novel uses of peptides derived from the HIV gp41 fusion peptide domain, in methods for prevention or treatment of autoimmune and other T cell-mediated pathologies. These peptides were demonstrated to be effective at concentrations about 100 fold lower than the peptides of the invention by Manolios (U.S. Pat. No. 6,057,294; US Pat. Appl. 20050070478). However, the suggested peptide sequences of this invention are based on the only amino acid sequences, the primary sequence of the HIV gp41 fusion peptide domain, which in addition to the lack of the primary criteria for rational design of these immunomodulatory peptides and optimizing their immunomodulatory activity, strongly limits further optimization of efficacy and specificity of targeting and inhibiting TCR by peptide variants.
Yet still another prior art (US Pat. Appl. 20080096809) provides membrane binding diastereomeric peptides comprising amino acid sequences corresponding to a fragment of a transmembrane proteins, wherein at least two amino acid residues of the diastereomeric peptides being in a D-isomer configuration. These peptides are suggested to be useful in inhibiting fusion membrane protein events, including specifically viral replication and transmission. However, these peptides are not designed specifically to treat T cell-related disorders.
What is needed in the art is a broad-based TCR-targeted therapy rationally designed to disrupt protein-protein interactions as specifically and effectively as viruses do that may be administered to treat various diseases having an underlying T cell etiology that is safe and effective.